Bacillus mHKcel cellulase

ABSTRACT

The present invention provides a novel cellulase nucleic acid sequence, designated mHKcel, and the corresponding mHKcel amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding mHKcel, recombinant mHKcel proteins and methods for producing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/467,315 filed Apr. 30, 2003, which is herein incorporated in itsentirety by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

This invention relates to a novel cellulase referred to herein asmHKcel. Also described are nucleic acids encoding the cellulase,compositions comprising said cellulase, methods of identifying novelcellulases and methods of using said compositions. Preferably thecellulase(s) are isolated from Bacillus species, preferably B.agaradhaerens. The present invention further relates to the use of thenovel cellulase in compositions recognized in the art as advantageouslyhaving cellulase added thereto, including, as an additive in a detergentcomposition, in the treatment of cellulose containing fabrics, in thetreatment of pulp and paper and in the treatment of starch for theproduction of high fructose corn-syrup or ethanol.

BACKGROUND OF THE INVENTION

Cellulose and hemicellulose are the most abundant plant materialsproduced by photosynthesis. They can be degraded and used as an energysource by numerous microorganisms, including bacteria, yeast and fungi,that produce extracellular enzymes capable of hydrolysis of thepolymeric substrates to monomeric sugars (Aro et al., 2001). As thelimits of non-renewable resources approach, the potential of celluloseto become a major renewable energy resource is enormous (Krishna et al.,2001). The effective utilization of cellulose through biologicalprocesses is one approach to overcoming the shortage of foods, feeds,and fuels (Ohmiya et al., 1997).

Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or betaD-glucosidic linkages) resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. Cellulases have beentraditionally divided into three major classes: endoglucanases (EC3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91)(“CBH”) and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC3.2.1.21) (“BG”). (Knowles et al., 1987; Shulein, 1988). Endoglucanasesact mainly on the amorphous parts of the cellulose fibre, whereascellobiohydrolases are also able to degrade crystalline cellulose(Nevalainen and Penttila, 1995). Thus, the presence of acellobiohydrolase in a cellulase system is required for efficientsolubilization of crystalline cellulose (Suurnakki, et al. 2000).Beta-glucosidase acts to liberate D-glucose units from cellobiose,cello-oligosaccharides, and other glucosides (Freer, 1993).

In order to efficiently convert crystalline cellulose to glucose thecomplete cellulase system comprising components from each of the CBH, EGand BG classifications is required, with isolated components lesseffective in hydrolyzing crystalline cellulose (Filho et al., 1996). Asynergistic relationship has been observed between cellulase componentsfrom different classifications. In particular, the EG-type cellulasesand CBH-type cellulases synergistically interact to more efficientlydegrade cellulose. See, e.g., Wood, 1985.

Although cellulase compositions have been previously described, thereremains a need for new and improved cellulase compositions for use inhousehold detergents, stonewashing compositions or laundry detergents,etc. Cellulases that exhibit improved performance are of particularinterest.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel cellulasehaving beneficial properties for use in detergents, treating textiles,biomass conversion and pulp and paper manufacturing.

It is an object of the present invention to provide polypeptides havingcellulolytic activity and polynucleotides encoding the polypeptides. Thepolypeptides may improve the degradation of cell wall material, e.g.,cellulose and/or hemicellulose. The polypeptides may also improve thestability or activity of other enzymes involved in the degradation ofplant cell wall material, e.g., biomass.

An object of the present invention is to provide a novel cellulase andderivatives thereof, methods of producing such cellulases, andcompositions comprising such novel cellulases. The present inventionfurther relates to the use of the novel cellulase and derivativesthereof in compositions recognized in the art as advantageously havingcellulase added thereto, including, as an additive in a detergentcomposition, in the treatment of textiles such as cellulose-containingfabrics and fibers useful therefor, as an animal feed additive, inbiomass conversion, in the treatment of pulp and paper and in thetreatment of starch for the production of high fructose corn-syrup orethanol.

It is a further object of the present invention to provide for a methodof producing a novel cellulase via heterologous expression fromrecombinant host cells.

It is yet a further object of the present invention to provide a nucleicacid sequence encoding the inventive cellulase. In one aspect, thenucleic acid and amino acid sequence facilitate commercial production ofthe novel cellulase and cellulase compositions of the invention.

It is still a further object of the present invention to provide a novelcellulase having excellent properties for use in detergents, treatingtextiles, as a feed supplement and in pulp and paper manufacturing. In afurther aspect, the cellulase finds use in biomass conversion.

In a first aspect, the invention includes an isolated polynucleotidehaving a sequence which encodes mHKcel, a sequence complementary to themHKcel gene coding sequence, and a composition comprising thepolynucleotide. The polynucleotide may be mRNA, DNA, cDNA, genomic DNA,or an antisense analog thereof.

In one embodiment, a mHKcel polynucleotide may comprise an isolatednucleic acid molecule which hybridizes to the complement of the nucleicacid presented as SEQ ID NO:2 under moderate to high stringencyconditions, where the nucleic acid molecule encodes a mHKcel polypeptidethat exhibits cellulose binding activity.

The polynucleotide having at least 80%, 85%, 90%, 95%, 98% or moresequence identity to the sequence presented as SEQ ID NO:2 may encode amHKcel protein. In a specific embodiment, the polynucleotide comprises asequence substantially identical to SEQ ID NO:2. The invention alsocontemplates fragments of the polynucleotide, preferably at least about15-30 nucleotides in length.

In a second aspect, a novel cellulase or a derivative is provided whichis obtainable from a Bacillus. Preferably, the cellulase of theinvention comprises an amino acid sequence according to FIG. 3 (SEQ IDNO:3), a fragment, or a derivative thereof, having greater than 90%sequence identity, preferably greater than 95% sequence identity andmore preferably greater than 97% sequence identity to an active portionthereto.

In a third aspect the present invention relates to a nucleic acidconstruct comprising the nucleotide sequence, which encodes for thepolypeptide of the invention, operably linked to one or more controlsequences that direct the production of the polypeptide in a suitablehost.

The invention further provides recombinant expression vectors containinga nucleic acid sequence encoding mHKcel or a fragment or splice variantthereof, operably linked to regulatory elements effective for expressionof the protein in a selected host In a related aspect, the inventionincludes a host cell containing the vector.

In a fourth aspect the present invention relates to a recombinantexpression vector comprising the nucleic acid construct of theinvention.

In a fifth aspect the present invention relates to a recombinant hostcell comprising the nucleic acid construct of the invention.

The invention further includes a method for producing mHKcel byrecombinant techniques, by culturing recombinant prokaryotic oreukaryotic host cells comprising nucleic acid sequence encoding mHKcelunder conditions effective to promote expression of the protein, andsubsequent recovery of the protein from the host cell or the cellculture medium.

In a sixth aspect the present invention relates to a method forproducing a polypeptide of the invention, the method comprising: (a)cultivating a microorganism, which in its wild-type form is capable ofproducing the polypeptide, to produce the polypeptide; and (b)recovering the polypeptide.

In a seventh aspect the invention provides for an enzymatic compositionuseful in the conversion of cellulose to ethanol. In a preferredembodiment the enzymatic composition comprises mHKcel. The compositionmay further comprise additional cellulase enzymes such as endoglucanasesand/or cellbiohydrolases. The composition may be enriched in mHKcel.

In one embodiment the invention provides a method of identifying novelenzymes by isolating total microbial community DNA from an environment,constructing a genomic DNA library in E. coli, screening the library forexpression of cellulase activity, identifying the cellulase gene in thecellulase-positive clone arid characterising the novel cellulase enzyme.

Further provided herein are analytical methods for detecting mHKcelnucleic acids and mHKcel proteins also form part of the invention.

According to yet another embodiment of the invention, a method oftransforming a suitable microorganism with nucleic acid sequenceencoding a cellulase according to the invention is provided. A method ofproducing the cellulase according to the invention from said transformedmicroorganism is provided.

A further object of the invention is to provide an expression vectorparticularly effective in Streptomyces. Streptomyces serve as alternatehost cells for the production of various proteins and with respect tothe expression and production of cellulases may offer a number ofadvantages over Bacillus host cells particularly when cells are grown ata high cell density. A preferred expression vector comprises aregulatory polynucleotide sequence including a promoter sequence derivedfrom a glucose isomerase gene of Actinoplanes, a signal sequence derivedfrom a Streptomyces cellulase gene, and a DNA sequence encoding acellulase, particularly a cellulase according to the invention.

In a preferred embodiment of the present invention, a full-lengthcellulase is obtainable from Bacillus.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the scope and spirit of the invention will becomeapparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the environmental nucleotide sequence (SEQ ID NO:1).

FIG. 2 illustrates a nucleic acid sequence encoding the novel cellulase(SEQ ID NO:2).

FIG. 3 illustrates the deduced amino acid sequence of the inventivecellulase (SEQ ID NO:3).

FIG. 4 is a graph depicting the enzyme activity with increasing sodiumchloride concentration.

FIG. 5 is a graph depicting the influence of pH on mHKcel activity overthe pH range of 7-12.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel FM et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

All publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

I. Definitions

“Cellulase,” “cellulolytic enzymes” or “cellulase enzymes” means theinventive bacterial endoglucanase described herein. Three differenttypes of cellulase enzymes act synergistically to convert cellulose andits derivatives to glucose.

The term “cellulase” refers to a category of enzymes capable ofhydrolyzing cellulose polymers to shorter cello-oligosaccharideoligomers, cellobiose and/or glucose. Numerous examples of cellulases,such as exoglucanases, exocellobiohydrolases, endoglucanases, andglucosidases have been obtained from cellulolytic organisms,particularly including fungi, and bacteria. The enzymes made by thesemicrobes are mixtures of proteins with three types of actions useful inthe conversion of cellulose to glucose: endoglucanases (EG),cellobiohydrolases (CBH), and beta-glucosidase. These three differenttypes of cellulase enzymes act synergistically to convert cellulose andits derivatives to glucose.

Many microbes make enzymes that hydrolyze cellulose, including the woodrotting fungus Trichoderma, the compost bacteria Thermomonospora,Bacillus, and Cellulomonas; Streptomyces; and the fungi Humicola,Aspergillus and Fusarium.

By the term “host cell” is meant a cell that contains a vector andsupports the replication, and/or transcription or transcription andtranslation (expression) of the expression construct. Host cells for usein the present invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as yeast, plant, insect, amphibian, or mammaliancells. In a one embodiment according to the present invention, “hostcell” means the cells of the genus Bacillus. In another preferredembodiment according to the invention, “host cell” means the cells ofStreptomyces. A Streptomyces means any bacterial strain that is a memberof the genus Streptomyces as classified in Buchanan et al., The ShorterBergey's Manual For Determinative Bacteriology (Williams & Wilkens1982). Particularly preferred strains of Streptomyces include S.lividens, S. rubiginosus, and S. coelicolor. S. lividens is described inLomovskaya et al., J. Virology 9:258 (1972). However, one of skill willrealize that any appropriate host cell, e.g., bacterial, fungal,eukaryotic and plant cell may be used.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide” or “secretory signal peptide”)that, as a component of a larger polypeptide, directs the largerpolypeptide through a secretory pathway of a cell in which it issynthesized. The larger peptide is commonly cleaved to remove thesecretory peptide during transit through the secretory pathway to yieldthe secretory signal peptide and a smaller peptide commonly referred toas the mature polypeptide.

As used herein, the phrases “whole cellulase preparation” and “wholecellulase composition” are used interchangeably and refer to bothnaturally occurring and non-naturally occurring compositions. A“naturally occurring” composition is one produced by a naturallyoccurring source and which comprises, for example, one or morecellobiohydrolase-type, one or more endoglucanase-type, and one or moreβ-glucosidase components wherein each of these components is found atthe ratio produced by the source. Certain fungi produce completecellulase systems which include exo-cellobiohydrolases or CBH-typecellulases, endoglucanases or EG-type cellulases and beta-glucosidasesor BG-type cellulases (Schulein, 1988). However, sometimes these systemslack CBH-type cellulases and bacterial cellulases also typically includelittle or no CBH-type cellulases. A naturally occurring composition isone that is produced by an organism unmodified with respect to thecellulolytic enzymes such that the ratio of the component enzymes isunaltered from that produced by the native organism.

A “non-naturally occurring” composition encompasses those compositionsproduced by: (1) combining component cellulolytic enzymes either in anaturally occurring ratio or non-naturally occurring, i.e., altered,ratio; or (2) modifying an organism to overexpress or underexpress oneor more cellulolytic enzyme; or (3) modifying an organism such that atleast one cellulolytic enzyme is deleted or (4) modifying an organism toexpress a heterologous component cellulolytic enzyme.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. The promoter may bethe promoter normally associated with the downstream gene or it may beheterologous, i.e., from another gene or another microorganism as longas it function to direct the gene. A preferred promoter when thetransformation host cell is Bacillus is the aprE promoter. In one aspectthe promoter is an inducible promoter. In one aspect, when the host cellis a filamentous fungus, the promoter is the T. reesei cbh1 promoterwhich is deposited in GenBank under Accession Number D86235. In anotheraspect the promoter is a cbh II or xylanase promoter from T. reesei.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader, i.e., a signal peptide, is operably linkedto DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice.

“DNA construct” or “DNA vector” means a nucleotide sequence whichcomprises one or more DNA fragments encoding the novel cellulase.Included in “DNA vectors” are “expression vectors.” Typical expressionvectors contain regulatory sequences such as, transcription andtranslation terminators, transcription and translation initiationsequences, signal sequences, and promoters useful for regulation of theexpression of the particular nucleic acid. The term “promoter” is usedin its ordinary sense to refer to a polynucleotide sequence involved inthe control of the initiation of transcription of a polynucleotidesequence encoding a protein. A “signal sequence” refers to a signalpeptide or a portion of a protein that is capable of directing thetransport of a desired protein in bioactive form from a host. The matureform of an extracellular protein lacks the signal sequence which iscleaved off during the secretion process. While not meant to limit theinvention, the number of amino acid residues in a signal peptide may bebetween about 5 and about 100 amino acid residues. Signal sequence maybe modified to provide for cloning sites that allow for the ligation ofDNA or insertion of DNA encoding a cellulase. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in prokaryotes, eukaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems.Vectors are suitable for replication and integration in prokaryotes,eukaryotes, or both. See, Giliman and Smith, Gene 8:81-97 (1979);Roberts et al., Nature 328:731-734 (1987); Berger and Kimmel, GUIDETOMOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, VOL 152, AcademicPress, Inc., San Diego, Calif. (“Berger”); Scheider, B., et al., ProteinExpr. Purif 6435:10 (1995); Sambrook et al. MOLECULAR CLONING—ALABORATORY MANUAL (2ND ED.) VOL. 1-3, Cold Springs Harbor Publishing(1989) (“Sambrook”); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubelet al. (eds.), Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1997Supplement) (“Ausubel”). Cloning vectors useful in Streptomyces areknown and reference is made to U.S. Pat. Nos. 4,338,397; 4,411,994;4,513,085; 4,513,086; 4,745,056; 5,514,590; and 5,622,866 andWO88/07079.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

In the present context, the term “substantially pure polypeptide” meansa polypeptide preparation which contains at the most 10% by weight ofother polypeptide material with which it is natively associated (lowerpercentages of other polypeptide material are preferred, e.g. at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most ½% by weight). Thus, it ispreferred that the substantially pure polypeptide is at least 92% pure,i.e. that the polypeptide constitutes at least 92% by weight of thetotal polypeptide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polypeptidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides disclosed herein arein “essentially pure form”, i.e. that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyassociated. This can be accomplished, for example, by preparing thepolypeptide by means of well-known recombinant methods. Herein, the term“substantially pure polypeptide” is synonymous with the terms “isolatedpolypeptide” and “polypeptide in isolated form”.

In general, nucleic acid molecules which encode the mHKcel willhybridize, under moderate to high stringency conditions to the sequenceprovided herein as SEQ ID NO:2 (the mHKcel). However, in some cases amHKcel-encoding nucleotide sequence is employed that possesses asubstantially different codon usage, while the protein encoded by themHKcel-encoding nucleotide sequence has the same or substantially thesame amino acid sequence as the native protein. For example, the codingsequence may be modified to facilitate faster expression of mHKcel in aparticular prokaryotic or eukaryotic expression system, in accordancewith the frequency with which a particular codon is utilized by thehost. Te'o, et al. (2000), for example, describes the optimization ofgenes for expression in filamentous fungi.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm−5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “moderate” or “intermediate stringency” at about 10-20° below the Tmof the probe; and “low stringency” at about 20-25° below the Tm.Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are-used toidentify sequences having about 80% or more sequence identity with theprobe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al., 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5× Denhardt's solution, 0.5%SDS and 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

It follows that the term “mHKcel expression” refers to transcription andtranslation of the mHKcel cellulase gene, the products of which includeprecursor RNA, mRNA, polypeptide, post-translationally processedpolypeptides. By way of example, assays for mHKcel expression includeWestern blot for mHKcel protein, Northern blot analysis and reversetranscriptase polymerase chain reaction (RT-PCR) assays for mHKcel mRNA,and endoglucanase activity assays as described in Shoemaker S. P. andBrown R. D. Jr. (Biochim. Biophys. Acta, 1978, 523:133-146) and Schulein(1988).

As used herein, the term “surfactant” refers to any compound generallyrecognized in the art as having surface active qualities. Thus, forexample, surfactants comprise anionic, cationic and nonionic surfactantssuch as those commonly found in detergents. Anionic surfactants includelinear or branched alkylbenzenesulfonates; alkyl or alkenyl ethersulfates having linear or branched alkyl groups or alkenyl groups; alkylor alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholyticsurfactants include quatemary ammonium salt sulfonates, and betaine-typeampholytic surfactants. Such ampholytic surfactants have both thepositive and negative charged groups in the same molecule. Nonionicsurfactants may comprise polyoxyalkylene ethers, as well as higher fattyacid alkanolamides or alkylene oxide adduct thereof, fatty acidglycerine monoesters, and the like.

As used herein, the term “cellulose containing fabric” refers to anysewn or unsewn fabrics, yarns or fibers made of cotton or non-cottoncontaining cellulose or cotton or non-cotton containing cellulose blendsincluding natural cellulosics and manmade cellulosics (such as jute,flax, ramie, rayon, and lyocell).

As used herein, the term “cotton-containing fabric” refers to sewn orunsewn fabrics, yarns or fibers made of pure cotton or cotton blendsincluding cotton woven fabrics, cotton knits, cotton denims, cottonyarns, raw cotton and the like.

As used herein, the term “stonewashing composition” refers to aformulation for use in stonewashing cellulose containing fabrics.Stonewashing compositions are used to modify cellulose containingfabrics prior to sale, i.e., during the manufacturing process. Incontrast, detergent compositions are intended for the cleaning of soiledgarments and are not used during the manufacturing process.

As used herein, the term “detergent composition” refers to a mixturewhich is intended for use in a wash medium for the laundering of soiledcellulose containing fabrics. In the context of the present invention,such compositions may include, in addition to cellulases andsurfactants, additional hydrolytic enzymes, builders, bleaching agents,bleach activators, bluing agents and fluorescent dyes, cakinginhibitors, masking agents, cellulase activators, antioxidants, andsolubilizers.

As used herein, the terms “active” and “biologically active” refer to abiological activity associated with a particular protein and are usedinterchangeably herein. For example, the enzymatic activity associatedwith a protease is proteolysis and, thus, an active protease hasproteolytic activity. It follows that the biological activity of a givenprotein refers to any biological activity typically attributed to thatprotein by those of skill in the art.

When employed in enzymatic solutions, the mHKcel component is generallyadded in an amount sufficient to allow the highest rate of release ofsoluble sugars from the biomass. The amount of mHKcel component addeddepends upon the type of biomass to be saccharified which can be readilydetermined by the skilled artisan. However, when employed, the weightpercent of the mHKcel component relative to any other cellulase typecomponents present in the cellulase composition is from preferably about1, preferably about 5, preferably about 10, preferably about 15, orpreferably about 20 weight percent to preferably about 25, preferablyabout 30, preferably about 35, preferably about 40, preferably about 45or preferably about 50 weight percent. Furthermore, preferred ranges maybe about 0.5 to about 15 weight percent, about 0.5 to about 20 weightpercent, from about 1 to about 10 weight percent, from about 1 to about15 weight percent, from about 1 to about 20 weight percent, from about 1to about 25 weight percent, from about 5 to about 20 weight percent,from about 5 to about 25 weight percent, from about 5 to about 30 weightpercent, from about 5 to about 35 weight percent, from about 5 to about40 weight percent, from about 5 to about 45 weight percent, from about 5to about 50 weight percent, from about 10 to about 20 weight percent,from about 10 to about 25 weight percent, from about 10 to about 30weight percent, from about 10 to about 35 weight percent, from about 10to about 40 weight percent, from about 10 to about 45 weight percent,from about 10 to about 50 weight percent, from about 15 to about 20weight percent, from about 15 to about 25 weight percent, from about 15to about 30 weight percent, from about 15 to about 35 weight percent,from about 15 to about 30 weight percent, from about 15 to about 45weight percent, from about 15 to about 50 weight percent.

II. Molecular Biology

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994)).

To obtain high level expression of a cloned gene, the heterologous geneis preferably positioned about the same distance from the promoter as isin the naturally occurring cellulase gene. As is known in the art,however, some variation in this distance can be accommodated withoutloss of promoter function.

Those skilled in the art are aware that a natural promoter can bemodified by replacement, substitution, addition or elimination of one ormore nucleotides without changing its function. The practice of theinvention encompasses and is not constrained by such alterations to thepromoter.

The expression vector/construct typically contains a transcription unitor expression cassette that contains all the additional elementsrequired for the expression of the heterologous sequence. A typicalexpression cassette thus contains a promoter operably linked to theheterologous nucleic acid sequence and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

The practice of the invention is not constrained by the choice ofpromoter in the genetic construct. The only constraint on the choice ofpromoter is that it is functional in the host cell used. A preferredpromoter when the transformation host cell is Bacillus is the aprEpromoter.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includebacteriophages λ and M13, as well as plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST,and LacZ. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, e.g., c-myc.

The elements that are typically included in expression vectors alsoinclude a replicon, a gene encoding antibiotic resistance to permitselection of bacteria that harbor recombinant plasmids, and uniquerestriction sites in nonessential regions of the plasmid to allowinsertion of heterologous sequences. The particular antibioticresistance gene chosen is not critical, any of the many resistance genesknown in the art are suitable.

The methods of transformation of the present invention may result in thestable integration of all or part of the transformation vector into thegenome of the filamentous fungus. However, transformation resulting inthe maintenance of a self-replicating extra-chromosomal transformationvector is also contemplated.

The gene encoding the cellulase of the present invention can be clonedusing λ-phage (expression) vectors and E. coli host cells.(Alternatively PCR cloning using consensus primers designed on conserveddomains may be used.) Applicants have discovered that transformation ofthe gene encoding the cellulase of the present invention and expressionin E. coli results in an active protein. After a first cloning step inE. coli, a cellulase gene according to the present invention can betransferred to a more preferred industrial expression host such asBacillus or Streptomyces species, a filamentous fungus such asAspergillus or Trichoderma, or a yeast such as Saccharomyces. High levelexpression and secretion obtainable in these host organisms allowsaccumulation of the cellulase in the fermentation medium from which itcan subsequently be recovered.

A preferred general transformation and expression protocol for proteasedeleted Bacillus strains is provided in Ferrari et al., U.S. Pat. No.5,264,366, incorporated herein by reference. Transformation andexpression in Aspergillus is described in, for example, Berka et al.,U.S. Pat. No. 5,364,770, incorporated herein by reference.

Many standard transfection methods can be used to produce Trichodermareesei cell lines that express large quantities of the heterologusprotein. Some of the published methods for the introduction of DNAconstructs into cellulase-producing strains of Trichoderma includeLorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356;Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174;Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6:155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl.Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila andKolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, forStreptomyces Hopwood et al., 1985, The John Innes Foundation, Norwich,UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi,1990, FEMS Microbiol. Lett. 55: 135-138), all incorporated herein byreference.

However, any of the well-known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, biolistics, liposomes, microinjection, plasma vectors,viral vectors and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). Also ofuse is the Agrobacterium-mediated transfection method described in U.S.Pat. No. 6,255,115. It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one gene into the host cell capable of expressing the heterologousgene.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofgenes under control of cellulase gene promoter sequences. Large batchesof transformed cells can be cultured as described below. Finally,product is recovered from the culture using standard techniques.

Thus, the invention herein provides for the expression and enhancedsecretion of the inventive cellulases whose expression is under controlof cellulase gene promoter sequences including naturally occurringcellulase genes, fusion DNA sequences, and various heterologousconstructs. The invention also provides processes for expressing andsecreting high levels of the inventive cellulases.

III. Identification of Nucleic Acids and Encoded Protein Sequences

A genomic library from Bacillus agaradhaerans (DSM 8721) was preparedusing standard techniques known in the art. This organism produces analkaline cellulase, (endo-1,4beta-glucanase), belonging to cellulasefamily 5 of glycosyl hydrolases, endoglucanase 5A, EC 3.2.1.4,Swiss-Prot: 085465, entry name GUN5_BACAG. EBI accession numberAF067428) the gene for which is 1203 bp in length, (Davies et al. 1998).Cellulase positive clones were detected with an incidence of 1/3000 inthe plate assay. In the process for isolating a gene according to anaspect of the present invention, degenerate primers based on the codingsequence for this enzyme were used. Unexpectedly, however, no PCRproduct was obtained using primers known to amplify the known B.agaradhaerans cellulase. The complete sequence of the insert coding forthe cellulase was therefore determined by primer walking.

The process for isolating a gene according to the second aspect of thepresent invention makes use of its homology to a nucleotide sequencecomprising all or part of the nucleotide sequence of SEQ ID NO:2 asshown in the sequence listing. Examples of such processes include:

-   -   a) screening a gene library which presumably contains a mHKcel        gene using the nucleotide sequence as a probe.    -   b) preparing a primer based on the nucleotide sequence        information, then performing PCR using a sample which presumably        contains a mHKcel gene as a template.

More specifically, process a) above comprises:

-   -   a) preparing a gene library which presumably contains a        cellulase gene, screening the gene library using a nucleotide        sequence comprising all or part of the nucleotide sequence of        SEQ ID NO:2 as shown in the sequence listing to select sequences        which hybridize with the nucleotide sequence comprising all or        part of the nucleotide sequence of SEQ ID NO:2 as shown in the        sequence listing from the gene library, then isolating the        selected sequences, and isolating a mHKcel gene from the        sequences which have been selected and isolated from the gene        library.

The gene library may be a genomic DNA library or a cDNA library, and maybe prepared according to a known procedure.

IV. Protein Expression

Proteins of the present invention are produced by culturing cellstransformed with an expression vector containing the inventive cellulasegene whose expression is under control of promoter sequences. Thepresent invention is particularly useful for enhancing the intracellularand/or extracellular production of proteins. The protein may behomologous or heterologous.

Proteins of the present invention may also be modified in a way to formchimeric molecules comprising a protein of interest fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the protein of interest with atag polypeptide which provides an epitope to which an anti-tag antibodycan selectively bind. The epitope tag is generally placed at theamino-or carboxyl-terminus of the protein of interest.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelationtags, the flu HA tag polypeptide and its antibody 12CA5 (Field et al.,Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular andCellular Biology 5:3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6):547-553 (1990)). Other tag polypeptides include theFLAG-peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3epitope peptide (Martin et al., Science 255:192-194 (1992)); tubulinepitope peptide (Skinner et al., J. Biol. Chem. 266:15163-15166 (1991));and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc.Natl. Acad. Sci. USA 87:6393-6397 (1990)).

Conditions appropriate for expression of said mHKcel gene comprisesproviding to the culture the components necessary for growth and/orexpression of the inventive cellulase. Optimal conditions for theproduction of the proteins will vary with the choice of the host cell,and with the choice of protein to be expressed. Such conditions will beeasily ascertained by one skilled in the art through routineexperimentation or optimization.

The protein of is typically purified or isolated after expression. Theprotien of interest may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the protein of interest may be purified using a standard anti-protein ofinterest antibody column. Ultrafiltration and diafiltration techniques,in conjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, ProteinPurification (1982); The degree of purification necessary will varydepending on the use of the protein of interest. In some instances nopurification will be necessary.

V. Utility of Cellulase

Treatment of textiles according to the present invention contemplatestextile processing or cleaning with a composition comprising thecellulase of this invention. Such treating includes, but is not limitedto, stonewashing, modifying the texture, feel and/or appearance ofcellulose-containing fabrics or other techniques used duringmanufacturing or cleaning/reconditioning of cellulose-containingfabrics. Additionally, treating within the context of this inventioncontemplates the removal of “immature” or “dead” cotton from cellulosicfabric or fibers. Immature cotton is significantly more amorphous thanmature cotton and because of, for example, uneven dyeing. Thecomposition contemplated in the present invention further includes acellulase component for use in washing a soiled manufacturedcellulose-containing fabric. For example, a cellulase of this inventionmay be used in a detergent composition for washing laundry. Detergentcompositions useful in accordance with the present invention includespecial formulations such as pre-wash, pre-soak and home-use colorrestoration compositions. Such treating compositions, as describedherein, may be in the form of a concentrate which requires dilution orin the form of a dilute solution or a form which can be applied directlyto the cellulose-containing fabric. General treatment techniques forcellulase treatment of textiles are described in, for example, EPPublication No. 220 016 and GB Application Nos. 1,368,599 and 2,095,275.

Treatment of a cellulosic material according to the present inventionfurther contemplates the treatment of animal feed, pulp and/or paper,food and grain for purposes known in the art. For example, cellulasesare known to increase the value of animal feed, improve the drainabilityof wood pulp, enhance food products and reduce fiber in grain during thegrain wet milling process or dry milling process.

Treating according to the instant invention comprises preparing anaqueous solution which contains an effective amount of a cellulase or acombination of cellulases together with other optional ingredientsincluding, for example, a buffer, a surfactant, and/or a scouring agent.An effective amount of a cellulase enzyme composition is a concentrationof cellulase enzyme sufficient for its intended purpose. Thus, forexample, an “effective amount” of cellulase in a stonewashingcomposition according to the present invention is that amount which willprovide the desired effect, e.g., to produce a worn and faded look inseams and on fabric panels. Similarly, an “effective amount” ofcellulase in a composition intended for improving the feel and/orappearance of a cellulose-containing fabric is the amount that producesmeasurable improvements in the feel, e.g., improving the smoothness ofthe fabric, or appearance, e.g., removing pills and fibrils which tendto reduce the sharpness in appearance of a fabric. The amount ofcellulase employed is also dependent on the equipment employed, theprocess parameters employed (the temperature of the cellulase treatmentsolution, the exposure time to the cellulase solution, and the like),and the cellulase activity (e.g., a particular solution will require alower concentration of cellulase where a more active cellulasecomposition is used as compared to a less active cellulase composition).The exact concentration of cellulase in the aqueous treatment solutionto which the fabric to be treated is added can be readily determined bythe skilled artisan based on the above factors as well as the desiredresult. In stonewashing processes, it has generally been preferred thatthe cellulase be present in the aqueous treating solution in aconcentration of from about 0.5 to 5,000 ppm and most preferably about10 to 200 ppm total protein. In compositions for the improvement of feeland/or appearance of a cellulose-containing fabric, it has generallybeen preferred that the cellulase be present in the aqueous treatingsolution in a concentration of from about 0.1 to 2000 ppm and mostpreferably about 0.5 to 200 ppm total protein.

In a preferred treating embodiment, a buffer is employed in the treatingcomposition such that the concentration of buffer is sufficient tomaintain the pH of the solution within the range wherein the employedcellulase exhibits activity. The pH at which the cellulase exhibitsactivity depends on the nature of the cellulase employed. The exactconcentration of buffer employed will depend on several factors whichthe skilled artisan can readily take into account. For example, in apreferred embodiment, the buffer as well as the buffer concentration areselected so as to maintain the pH of the final cellulase solution withinthe pH range required for optimal cellulase activity. The determinationof the optimal pH range of the cellulases of the invention can beascertained according to well-known techniques. Suitable buffers at pHwithin the activity range of the cellulase are also well known to thoseskilled in the art in the field.

In addition to cellulase and a buffer, the treating composition mayoptionally contain a surfactant. Suitable surfactants include anysurfactant compatible with the cellulase being utilized and the fabricincluding, for example, anionic, non-ionic and ampholytic surfactants.Suitable anionic surfactants include, but are not limited to, linear orbranched alkylbenzenesulfonates; alkyl or alkenyl ether sulfates havinglinear or branched alkyl groups or alkenyl groups; alkyl or alkenylsulfates; olefinsulfonates; alkanesulfonates and the like. Suitablecounter ions for anionic surfactants include, but are not limited to,alkali metal ions such as sodium and potassium; alkaline earth metalions such as calcium and magnesium; ammonium ion; and alkanolamineshaving 1 to 3 alkanol groups of carbon number 2 or 3. Ampholyticsurfactants include, e.g., quaternary ammonium salt sulfonates, andbetaine-type ampholytic surfactants. Such ampholytic surfactants haveboth the positive and negative charged groups in the same molecule.Nonionic surfactants generally comprise polyoxyalkylene ethers, as wellas higher fatty acid alkanolamides or alkylene oxide adduct thereof, andfatty acid glycerine monoesters. Mixtures of surfactants can also beemployed in manners known to those skilled in the art.

A concentrated cellulase composition can be prepared for use in themethods described herein. Such concentrates contain concentrated amountsof the cellulase composition described above, buffer and surfactant,preferably in an aqueous solution. When so formulated, the cellulaseconcentrate can readily be diluted with water so as to quickly andaccurately prepare cellulase preparations having the requisiteconcentration of each constituent. When aqueous concentrates areformulated, these concentrates can be diluted so as to arrive at therequisite concentration of the components in the cellulase solution asindicated above. As is readily apparent, such cellulase concentratespermit facile formulation of the cellulase solutions as well as permitfeasible transportation of the composition to the location where it willbe used. The treating concentrate can be in any art-recognized form, forexample, liquid, emulsion, gel, or paste. Such forms are well known tothose skilled in the art.

When a solid cellulase concentrate is employed, the cellulasecomposition may be a granule, a powder, an agglomerate or a solid disk.The granules can be formulated so as to contain materials to reduce therate of dissolution of the granules into the wash medium. Such materialsand granules are disclosed in U.S. Pat. No. 5,254,283 which isincorporated herein by reference in its entirety.

Other materials can also be used with or placed in the cellulasecomposition of the present invention as desired, including stones,pumice, fillers, solvents, enzyme activators, and anti-redepositionagents depending on the eventual use of the composition.

By way of example, stonewashing methods will be described in detail,however, the parameters described are readily modified by the skilledartisan for other applications, i.e., improving the feel and/orappearance of a fabric. The cellulose-containing fabric is contactedwith the cellulase containing stonewashing composition containing aneffective amount of the cellulase by intermingling the treatingcomposition with the stonewashing composition, and thus bringing thecellulase enzyme into proximity with the fabric. Subsequently, theaqueous solution containing the cellulase and the fabric is agitated. Ifthe treating composition is an aqueous solution, the fabric may bedirectly soaked in the solution. Similarly, where the stonewashingcomposition is a concentrate, the concentrate is diluted into a waterbath with the cellulose-containing fabric. When the stonewashingcomposition is in a solid form, for example a pre-wash gel or solidstick, the stonewashing composition may be contacted by directlyapplying the composition to the fabric or to the wash liquor.

The cellulose-containing fabric is incubated with the stonewashingsolution under conditions effective to allow the enzymatic action toconfer a stonewashed appearance to the cellulose-containing fabric. Forexample, during stonewashing, the pH, liquor ratio, temperature andreaction time may be adjusted to optimize the conditions under which thestonewashing composition acts. “Effective conditions” necessarily refersto the pH, liquor ratio, and temperature which allow the cellulaseenzyme to react efficiently with cellulose-containing fabric, in thiscase to produce the stonewashed effect. It is within the skill of thosein the art to maximize conditions for using the stonewashingcompositions according to the present invention.

The liquor ratios during stonewashing, i.e., the ratio of weight ofstonewashing composition solution (i.e., the wash liquor) to the weightof fabric, employed herein is generally an amount sufficient to achievethe desired stonewashing effect in the denim fabric and is dependentupon the process used. Preferably, the liquor ratios are from about 4:1to about 50:1; more preferably from about 5:1 to about 20:1, and mostpreferably from about 10:1 to about 15:1.

Reaction temperatures during stonewashing with the present stonewashingcompositions are governed by two competing factors. Firstly, highertemperatures generally correspond to enhanced reaction kinetics, i.e.,faster reactions, which permit reduced reaction times as compared toreaction times required at lower temperatures. Accordingly, reactiontemperatures are generally at least about 10° C. and greater. Secondly,cellulase is a protein which loses activity beyond a given reactiontemperature, which temperature is dependent on the nature of thecellulase used. Thus, if the reaction temperature is permitted to go toohigh, the cellulolytic activity is lost as a result of the denaturing ofthe cellulase. While standard temperatures for cellulase usage in theart are generally in the range of 35° C. to 65° C., and these conditionswould also be expected to be suitable for the cellulase of theinvention, the optimal temperature conditions should be ascertainedaccording to well known techniques with respect to the specificcellulase used.

Reaction times are dependent on the specific conditions under which thestonewashing occurs. For example, pH, temperature and concentration ofcellulase will all affect the optimal reaction time. Generally, reactiontimes are from about 5 minutes to about 5 hours, and preferably fromabout 10 minutes to about 3 hours and, more preferably, from about 20minutes to about 1 hour.

According to yet another preferred embodiment of the present invention,the cellulase of the invention may be employed in a detergentcomposition. The detergent compositions according to the presentinvention are useful as pre-wash compositions, pre-soak compositions, orfor cleaning during the regular wash or rinse cycle. Preferably, thedetergent composition of the present invention comprises an effectiveamount of cellulase, a surfactant, and optionally includes otheringredients described below.

An effective amount of cellulase employed in the detergent compositionsof this invention is an amount sufficient to impart the desirableeffects known to be produced by cellulase on cellulose-containingfabrics, for example, depilling, softening, anti-pilling, surface fiberremoval, anti-graying and cleaning. Preferably, the cellulase in thedetergent composition is employed in a concentration of from about 10ppm to about 20,000 ppm of detergent.

The concentration of cellulase enzyme employed in the detergentcomposition is preferably selected so that upon dilution into a washmedium, the concentration of cellulase enzyme is in a range of about0.01 to about 1000 ppm, preferably from about 0.02 ppm to about 500 ppm,and most preferably from about 0.5 ppm to about 250 ppm total protein.The amount of cellulase enzyme employed in the detergent compositionwill depend on the extent to which the detergent will be diluted uponaddition to water so as to form a wash solution.

The detergent compositions of the present invention may be in any artrecognized form, for example, as a liquid, in granules, in emulsions, ingels, or in pastes. Such forms are well known to the skilled artisan.When a solid detergent composition is employed, the cellulase ispreferably formulated as granules. Preferably, the granules can beformulated so as to additionally contain a cellulase protecting agent.The granule can be formulated so as to contain materials to reduce therate of dissolution of the granule into the wash medium. Such materialsand granules are disclosed in U.S. Pat. No. 5,254,283 which isincorporated herein by reference in its entirety.

The detergent compositions of this invention employ a surface activeagent, i.e., surfactant, including anionic, non-ionic and ampholyticsurfactants well known for their use in detergent compositions.

Suitable anionic surfactants for use in the detergent composition ofthis invention include linear or branched alkylbenzenesulfonates; alkylor alkenyl ether sulfates having linear or branched alkyl groups oralkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates; andalkanesul-fonates. Suitable counter ions for anionic surfactants includealkali metal ions such as sodium and potassium; alkaline earth metalions such as calcium and magnesium; ammonium ion; and alkanolamineshaving 1 to 3 alkanol groups of carbon number 2 or 3. Ampholyticsurfactants include quaternary ammonium salt sulfonates, andbetaine-type ampholytic surfactants. Such ampholytic surfactants haveboth the positive and negative charged groups in the same molecule.Nonionic surfactants generally comprise polyoxyal-kylene ethers, as wellas higher fatty acid alkanolamides or alkylene oxide adduct thereof,fatty acid glycerine monoesters, and the like. Suitable surfactants foruse in this invention are disclosed in British Patent Application No. 2094 826 A, the disclosure of which is incorporated herein by reference.Mixtures of such surfactants can also be used. The surfactant or amixture of surfactants is generally employed in the detergentcompositions of this invention in an amount from about 1 weight percentto about 95 weight percent of the total detergent composition andpreferably from about 5 weight percent to about 45 weight percent of thetotal detergent composition. In addition to the cellulase compositionand the surfactant(s), the detergent compositions of this invention canoptionally contain one or more of the following components:

Hydrolases Except Cellulase

Suitable hydrolases include carboxylate ester hydrolase, thioesterhydrolase, phosphate monoester hydrolase, and phosphate diesterhydrolase which act on the ester bond; glycoside hydrolase which acts onglycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds;thioether hydrolase which acts on the ether bond; anda-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase,acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptidehydrolase which act on the peptide bond. Preferable among them arecarboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptidehydrolase. Suitable hydrolases include (1) proteases belonging topeptidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin,chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C,papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin,aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein,gastrisin, cathepsin D., bromelin, keratinase, chymotrypsin C, pepsin C,aspergillopeptidase B, urokinase, carboxypeptidase A and B, andaminopeptidase; (2) glycoside hydrolases (cellulase which is anessential ingredient is excluded from this group) α-amylase, β-amylase,gluco amylase, invertase, lysozyme, pectinase, chitinase, anddextranase. Preferably among them are α-amylase and β-amylase. Theyfunction in acid to neutral systems, but one which is obtained frombacteria exhibits high activity in an alkaline system; (3) carboxylateester hydrolase including carboxyl esterase, lipase, pectin esterase,and chlorophyllase. Especially effective among them is lipase.

The hydrolase other than cellulase is incorporated into the detergentcomposition as much as required according to the purpose. It shouldpreferably be incorporated in an amount of 0.001 to 5 weight percent,and more preferably 0.02 to 3 weight percent, in terms of purifiedprotein. This enzyme should be used in the form of granules made ofcrude enzyme alone or in combination with other components in thedetergent composition. Granules of crude enzyme are used in such anamount that the purified enzyme is 0.001 to 50 weight percent in thegranules. The granules are used in an amount of 0.002 to 20 andpreferably 0.1 to 10 weight percent. As with cellulases, these granulescan be formulated so as to contain an enzyme protecting agent and adissolution retardant material.

Cationic Surfactants and Long-Chain Fatty Acid Salts

Such cationic surfactants and long-chain fatty acid salts includesaturated or unsaturated fatty acid salts, alkyl or alkenyl ethercarboxylic acid salts, α-sulfofatty acid salts or esters, aminoacid-type surfactants, phosphate ester surfactants, quaternary ammoniumsalts including those having 3 to 4 alkyl substituents and up to 1phenyl substituted alkyl substituents. Suitable cationic surfactants andlong-chain fatty acid salts are disclosed in British Patent ApplicationNo. 2 094 826 A, the disclosure of which is incorporated herein byreference. The composition may contain from about 1 to about 20 weightpercent of such cationic surfactants and long-chain fatty acid salts.

Builders

A. Divalent Sequestering Agents

The composition may contain from about 0 to about 50 weight percent ofone or more builder components selected from the group consisting ofalkali metal salts and alkanolamine salts of the following compounds:phosphates, phosphonates, phosphonocarboxylates, salts of amino acids,aminopolyacetates high molecular electrolytes, non-dissociatingpolymers, salts of dicarboxylic acids, and aluminosilicate salts.Suitable divalent sequestering gents are disclosed in British PatentApplication No. 2 094 826 A, the disclosure of which is incorporatedherein by reference.

B. Alkalis or Inorganic Electrolytes

The composition may contain from about 1 to about 50 weight percent,preferably from about 5 to about 30 weight percent, based on thecomposition of one or more alkali metal salts of the following compoundsas the alkalis or inorganic electrolytes: silicates, carbonates andsulfates as well as organic alkalis such as triethanolamine,diethanolamine, monoethanolamine and triisopropanolamine.

Antiredeposition Agents

The composition may contain from about 0.1 to about 5 weight percent ofone or more of the following compounds as antiredeposition agents:polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone andcarboxymethylcellulose.

Among them, a combination of carboxymethyl-cellulose and/or polyethyleneglycol with the cellulase composition of the present invention providesfor an especially useful dirt removing composition.

Bleaching Agents

The use of the cellulase of the present invention in combination with ableaching agent such as potassium monopersulfate, sodium percarbonate,sodium perborate, sodium sulfate/hydrogen peroxide adduct and sodiumchloride/hydrogen peroxide adduct or/and a photo-sensitive bleaching dyesuch as zinc or aluminum salt of sulfonated phthalocyanine furtherimproves the detergenting effects. Similarly, bleaching agents andbleach catalysts as described in EP 684 304 may be used.

Bluing Agents and Fluorescent Dyes

Various bluing agents and fluorescent dyes may be incorporated in thecomposition, if necessary. Suitable bluing agents and fluorescent dyesare disclosed in British Patent Application No. 2 094 826 A, thedisclosure of which is incorporated herein by reference.

Caking Inhibitors

The following caking inhibitors may be incorporated in the powderydetergent: p-toluenesulfonic acid salts, xylenesulfonic acid salts,acetic acid salts, sulfosuccinic acid salts, talc, finely pulverizedsilica, amorphous silicas, clay, calcium silicate (such as Micro-Cell ofJohns Manville Co.), calcium carbonate and magnesium oxide.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene,4,4′-butylidenebis(6-tert-butyl-3-methylphenol),2,2′-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol,distyrenated cresol, monostyrenated phenol, distyrenated phenol and1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

The solubilizers include, for example, lower alcohols such as ethanol,benzenesulfonate salts, lower alkylbenzenesulfonate salts such asp-toluenesulfonate salts, glycols such as propylene glycol,acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acidamides, benzoate salts and urea.

The detergent composition of the present invention can be used in abroad pH range from acidic to alkaline pH. In a preferred embodiment,the detergent composition of the present invention can be used in mildlyacidic, neutral or alkaline detergent wash media having a pH of fromabove 5 to no more than about 12.

Aside from the above ingredients, perfumes, buffers, preservatives, dyesand the like can be used, if desired, with the detergent compositions ofthis invention. Such components are conventionally employed in amountsheretofore used in the art.

When a detergent base used in the present invention is in the form of apowder, it may be one which is prepared by any known preparation methodsincluding a spray-drying method and a granulation method. The detergentbase obtained particularly by the spray-drying method, agglomerationmethod, dry mixing method or non-tower route methods are preferred. Thedetergent base obtained by the spray-drying method is not restrictedwith respect to preparation conditions. The detergent base obtained bythe spray-drying method is hollow granules which are obtained byspraying an aqueous slurry of heat-resistant ingredients, such assurface active agents and builders, into a hot space. After thespray-drying, perfumes, enzymes, bleaching agents, inorganic alkalinebuilders may be added. With a highly dense, granular detergent baseobtained such as by the spray-drying-granulation or agglomerationmethod, various ingredients may also be added after the preparation ofthe base.

When the detergent base is a liquid, it may be either a homogeneoussolution or a nonhomogeneous dispersion. For removing the decompositionof carboxymethylcellulose by the cellulase in the detergent, it isdesirable that carboxymethylcellulose is granulated or coated before theincorporation in the composition.

The detergent compositions of this invention may be incubated withcellulose-containing fabric, for example soiled fabrics, in industrialand household uses at temperatures, reaction times and liquor ratiosconventionally employed in these environments.

Detergents according to the present invention may additionally beformulated as a pre-wash in the appropriate solution at an intermediatepH where sufficient activity exists to provide desired improvementssoftening, depilling, pilling prevention, surface fiber removal orcleaning. When the detergent composition is a pre-soak (e.g., pre-washor pre-treatment) composition, either as a liquid, spray, gel or pastecomposition, the cellulase enzyme is generally employed from about0.0001 to about 1 weight percent based on the total weight of thepre-soak or pre-treatment composition. In such compositions, asurfactant may optionally be employed and when employed, is generallypresent at a concentration of from about 0.005 to about 20 weightpercent based on the total weight of the pre-soak. The remainder of thecomposition comprises conventional components used in the pre-soak,i.e., diluent, buffers, other enzymes (proteases), and the like at theirconventional concentrations.

It is contemplated that compositions comprising cellulase enzymesdescribed herein can be used in home use as a stand alone compositionsuitable for restoring color to faded fabrics (see, for example, U.S.Pat. No. 4,738,682, which is incorporated herein by reference in itsentirety) as well as used in a spot-remover and for depilling andantipilling (pilling prevention).

The use of the cellulase according to the invention may be particularlyeffective in feed additives and in the processing of pulp and paper.These additional industrial applications are described in, for example,PCT Publication No. 95/16360 and Finnish Granted Patent No. 87372,respectively.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given with theunderstanding that they are being offered to illustrate the presentinvention and should not be construed in any way as limiting its scope.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Sample Collection and Processing

This example illustrates how to collect samples and process them toobtain sufficient DNA to create a cDNA library.

Samples of water (250 ml) were collected from the littoral zone ofSonachi (Crater) Lake, Kenya using a 250-ml stainless steel beakermounted on the end of a flexible extendible 1-m pole and placed insealable plastic containers (Whirlpak) for transport to the laboratoryat ambient temperature. The temperature of the surface waters was 28°C., with pH 10 and a conductivity of 7.23 mS cm⁻¹ (at 27° C.).

To collect the microbial flora, water (750) from Sonachi (Crater) Lake,Kenya was filtered on site (using a hand operated vacuum pump) through asequence of sterile membrane filters (47 mm diameter), composed ofcellulose nitrate or cellulose acetate, of decreasing pore size, untilall water flow stopped. The sequence of filters was 8 μm, 3 μm and 0.22μm. The individual membrane filters were placed immediately into 10 mlof cold, sterile cell stabilization buffer (TES) containing 10 mM TrisHCl, pH8.0; 1 mM EDTA and 5% w/v NaCl in 30 ml sterile plastic universaltubes and kept on ice in a refrigerated cool box until they could beprocessed further, usually within 4 hours of sampling. The microbialmaterial on the filters was dispersed by vigorous vortex mixing withsterile glass beads (5 ml) and the cells pelleted in microfuge tubes bycentrifugation at 13,000 g for 5 min. The microbial material wasaliquoted to the microfuge tubes in volumes estimated to contain theequivalent of 10⁸ to 10⁹ bacterial cells, giving a total of 12 tubes.The DNA was extracted using the GenomicPrep™ Cells and Tissue DNAisolation kit (Amersham Pharmacia Biotech, Piscataway, N.J., USA)following the manufacturer's instructions. Cells in each tube wereresuspended in 600 μl of the Cell Lysis Solution provided, and incubatedat 80° C. for 5 min to lyse the cells. Samples prepared by this methodare stable at room temperature for at least 18 months, and weretransported back to the laboratory in this form. DNA extraction wascompleted by RNase A treatment, protein precipitation and isopropanolprecipitation of the DNA following the manufacturer's protocol. Each DNApellet was dissolved in 100 μl sterile Tris buffer 10 mM pH 8.5.

DNA yield was estimated by running 5 μl samples on a 0.5% w/v agarosegel and comparing with known amounts of bacterial genomic DNA. Thesamples were pooled, giving a total of about 20 μg DNA. Since yieldswere low, the material was supplemented with about 30% extra materialextracted from the water samples which were collected at the same timeas the on-site material and stored at 4° C. in the laboratory untilrequired. This amount of DNA, about 30 μg, was the amount of startingmaterial that preliminary experiments had shown was needed to carry outthe trial and bulk restriction digestion and size fractionation to givesufficient material for library construction.

Example 2 Library Construction

The floowing example details how to prepare a DNA library for use inscreening and detection of novel sequences in E. coli.

-   Preparation of DNA

The pooled DNA was used for construction of the genomic DNA library. Thepurified DNA was partially digested with Sau3A1 to give an averagefragment size of about 5 kb. Restricted DNA was size fractionated byelectrophoresis on 0.5% agarose in TAE (0.04 M Tris-acetate, 0.001 MEDTA pH 8.0). Material in the 1.5 to 10 kb range was excised andreplaced in a well of the same size cut in an unused part of the agarosegel and concentrated to a narrow band by reversed electrical current.The DNA band was excised and DNA extracted using the QIAGEN (Crawley,UK) QIAEXII gel extraction kit, following the manufacturer's guidelines.The eluted DNA was precipitated with ethanol and resuspended in 10 mMTris HCl buffer, pH 8.5.

-   Preparation of Lambda Libraries

The restricted DNA was cloned into a Lambda vector using theZAP-Express™ vector kit (predigested with BamH1 and alkaline phosphatasetreated) and the Gigapak® III Gold packaging extract (Stratagene,Amsterdam, The Netherlands) following the manufacturer's protocol. Theprimary libraries were amplified as per protocol by plating aliquotscontaining ˜5×10⁴ pfu with host E. coli strain XL1-Blue MRF′ on 150 mmPetri dishes and eluting the phage in buffer. Amplified libraries werestored in 7% v/v dimethyl sulphoxide at −80° C. after freezing in liquidnitrogen. The total primary titre was 1.8×10⁶ pfu and afteramplification 6.8×10⁹ pfu ml⁻¹.

-   Assessment of Library Quality

The phagemid vector pBK-CMV was excised from the Lambda ZAP libraryusing ExAssist helper phage (Stratagene) as described by themanufacturer, and used to infect E.coli strain XLOLR. Plasmid-containingclones were isolated by plating on Luria-Bertani (LB) agar containing 50μg ml⁻¹ kanamycin. Blue:white screening in the presence of Xgal[5-bromo-4-chloro-3-indoyl-β-D-galactoside] and IPTG[isopropylthio-β-D-galactoside] was used to determine cloningefficiency. If no DNA has been cloned into the Lambda vector, theβ-galactosidase gene is expressed in the presence of the inducer IPTG,resulting in cleavage of the substrate analogue Xgal to produce a bluepigment in the colony. If however a fragment of the genomic DNA has beensuccessfully cloned into the Lambda vector it disrupts the gene so thatno enzyme is produced and the colony remains white. The ratio of blue towhite colonies therefore can be used to calculate the percentage ofclones containing an insert. For this library the blue:white screen gavea ratio of 7 blue to 286 white colonies, indicating that 97% of theclones contained an insert of the genomic DNA. Twenty four colonies wereselected at random and plasmid DNA prepared using the Wizard®Plus SVMiniprep DNA purification system (Promega UK, Southampton) Restrictionanalysis using Pst1 and HindIII which flank the BamH1 cloning sitefollowed by agarose gel electrophoresis was used to determine insertsizes. One clone out of the 24 was found to have no detectable insert.The rest had inserts ranging from 1.5 kb to 8.0 kb.

Example 3 Library screening for cellulases

DNA libraries in the pBK-CMV phagemid were screened for cellulaseactivity in a plate assay of the E. coli clones. To detect cellulaseactivity the genomic libraries were plated on LB agar containingkanamycin, 0.5% w/v carboxymethylcellulose (low viscosity sodium salt;Sigma, Poole, UK) and IPTG (15 μl of a 0.5 M solution spread on thesurface of the agar in a 7 cm diameter Petri dish). Following overnightgrowth at 37° C., the colonies were overlayed with 3 ml molten 0.7% w/vagarose dissolved in water which had been cooled to 50° C. After thishad set, the plates were flooded with 0.1% w/v Congo Red solution for 30minutes followed by 2 washes with 1 M NaCl. Positive clones exhibitingextracellular cellulase activity were surrounded by a yellow haloagainst a red background (R. Teather and P. J. Wood, Applied &Environmental Microbiology, 43: 770-780, 1982).

The screening of 110,000 E.coli pBK-CMV clones yielded 4 zones ofclearing indicating potential cellullase-producing colonies. Three ofthese were successfully recovered as cellulase-producing clones afterhomogenising the agar plug removed from the cleared zone, streaking outfor single colonies and confirming the phenotype by the Congo Red test.

Example 4 Characterisation of a Cellulase-positive Clone

Plasmid DNA was isolated from the three cellulase positive clones, andthe size of the inserts determined by restriction digestion as describedabove. All three had the same size (about 3.5 kb) and the same sizefragments after digestion as determined by gel electrophoresis. Thisindicated that all three isolates were identical, derived byamplification of a single clone. This was confirmed by the first roundof sequencing of the plasmid DNA (using primer sites in the pBKCMVplasmid). This was carried out by the Protein and Nucleic Acid ChemistryLaboratory at Leicester University, using the Perkin Elmer ‘BigDye’terminator chemistry and the model 377 ABI automated DNA sequencer.Complete coverage of the sequence was obtained by ‘primer walking’ fromboth the 5′ and 3′ ends of the insert. The sequence was edited usingApplied Biosystems multisequence editor Seqed™ version 1.0.3. Sequencewas assembled with programmes in the GCG Wisconsin Package, version10.2-UNIX, available at the University of Leicester. This identified aninsert of environmental DNA of 3796 nucleotide bases (FIG. 1).

Example 5 Identification of the Cellulase-gene

Possible Open Reading Frames (ORF) in the nucleotide sequence of theinserted environmental DNA of clone mHKcel were identified using the ORFFind facility of the MapDraw program (DNASTAR, Brighton, Mass., USA) orORF Search from the Vector NTI Suite of programs (InforMax®, NorthBethesda, Md., USA).

This identified an ORF composed of 1716 nucleotides corresponding to aprotein of 571 amino acids, starting at position 923 of the insertsequence and ending at position 2638. The sequence of this ORF wasexcised using EditSeq (DNASTAR) and examined by BLAST programs.

The nucleotide sequence of this ORF is shown in FIG. 2.

An examination of the nucleotide sequence using the BLASTn program,which compares a nucleotide query sequence against a non-redundantnucleotide sequence database, indicated identity with parts of theBacillus halodurans genome, in particular 68% identity with 1585nucleotide region corresponding with a endo-beta-1,4-glucanase(“cellulase B”) (GenBank accession AP001509).

An examination of the nucleotide sequence using the BLASTx program,which compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase indicated significant similarity to a number of bacterialendocellulases. The highest alignment score revealed 67% identity (372amino acids) to a 553 amino acid region of an endo-β-1,4-glucanase(cellulase B) of the facultative alkaliphilic bacterium Bacillushalodurans strain C125, an enzyme comprising 574 amino acids (protein idBAB04322, accession AP001509). This newly identified gene coded for aprotein of 571 amino acids with 67% identity to the Bacillus haloduranscellulase.

The translated protein composed of 571 amino acids is shown in FIG. 3.

Example 6 Enzyme Characterization

Influence of Salt

Cells of E.coli pBK-CMV containing the mHKcel gene were suspended in 5ml buffer (20 mM TRIS-HCl, pH8.0; 500 mM NaCl; 0.1 mM EDTA; 0.1% TritonX-100) and disrupted by sonication on ice. The sonicated extracts wereexamined by agar diffusion assay on carboxymethylcellulose (CMC) atdifferent NaCl concentrations. Sonicated extracts (100 μL) and 1 in 10dilutions were placed in wells punched in CMC-agar plates containingvarying amounts of NaCl. The plates were incubated at 37° C. for 16hours and the resulting clearing zones indicating cellulose hydrolysismeasured in millimetres. The results (FIG. 4) indicate that thecellulase mHKcel is active over the range 0-25% w/v NaCl, although theactivity at 25% w/v NaCl is only about 50% of the activity at 0% NaCl.

Influence of DH

The influence of pH on cellulase activity was investigated using thepH-gradient plate method described by Grant & Tindall (Isolation ofalkaliphilic bacteria, In: Microbial Growth and Survival in ExtremeEnvironments, Academic Press, London, 1980, pp. 27-36). An agar mediumcontaining CMC was poured to a depth of 1 cm in square Petri dishes andallowed to set. A uniform trough 1 cm wide was cut from one edge of theplate and agar containing 20% w/v Na₂CO_(3·)10H₂O and 0.2 M NaOH(prepared by mixing equal volumes of sterile 0.4 M NaOH/40% w/vNa₂CO_(3·)10H₂O and 4% w/v agar at 60° C.) was poured into the trough.The plates were developed at 37° C. overnight to allow a uniformgradient from pH 12 to pH 7 to form. To test the pH tolerance of themHKcel cellulase a narrow trough was cut through the (agar) gradient atright angles to the original trough and filled with 1 ml of sonicatedcell extract. The plates were allowed to develop overnight at 37° C. Theplates were treated with Congo Red for 30 minutes to visualize the zoneof cellulose hydrolysis. The results shown in FIG. 5 indicate thatmHKcel cellulase is active to about pH 11.5.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A purified cellulase comprising a sequence selected from the groupconsisting of: (a) an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence presented in FIG. 3 (SEQ ID NO: 3);(b) the amino acid sequence presented in FIG. 3 (SEQ ID NO: 3); (c) apurified biologically active fragment having cellulase activity of theamino acid sequence encoded by the nucleotide sequence presented as SEQID NO: 2, wherein the identity is determined by the CLUSTAL-W program inMacVector version 6.5, operated with default parameters, including anopen gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.
 2. A purified cellulase obtainable from a Bacillushaving the amino acid sequence encoded by the nucleotide sequencepresented in FIG. 2 (SEQ ID NO:2).
 3. A purified enzyme having cellulaseactivity prepared by a method comprising the steps of: (a) culturing ahost cell transformed with a vector comprising a nucleic acid moleculeencoding the cellulase according to claim 1 in a suitable culture mediumunder suitable conditions to produce the cellulase; (b) obtaining saidproduced cellulase.
 4. A detergent composition, said compositioncomprising a polypeptide having cellulase activity selected from thegroup consisting of: (a) an amino acid sequence having at least 95%sequence identity to the amino acid sequence presented in FIG. 3 (SEQ IDNO: 3); (b) the amino acid sequence presented in FIG. 3 (SEQ ID NO: 3);(c) a purified biologically active fragment having cellulase activity ofthe amino acid sequence encoded by the nucleotide sequence presented asSEQ ID NO: 2, wherein the identity is determined by the CLUSTAL-Wprogram in MacVector version 6.5, operated with default parameters,including an open gap penalty of 10.0, an extended gap penalty of 0.1,and a BLOSUM 30 similarity matrix.
 5. A detergent composition comprisinga surfactant and a cellulase according to claim
 1. 6. The detergentaccording to claim 4, wherein said detergent is a laundry detergent. 7.The detergent according to claim 4, wherein said detergent is a dishdetergent.